Structure and Dynamics of Meprin β in Complex with a Hydroxamate-Based Inhibitor

The astacin protease Meprin β represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin β inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin β holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors.     

IT-basierte Entscheidungsunterstützung am Beispiel der Subprimekrise: Wunderwaffe oder Teufelszeug?

Ohne Zusammenfassung     

Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.     

Stable isotope dilution analysis of the Fusarium mycotoxins deoxynivalenol and 3-acetyldeoxynivalenol

Trichothecenes are secondary metabolites produced by several fungi of the Fusarium genus during their growth period. They inhibit protein biosynthesis in eukaryotic cells resulting in numerous toxic effects such as diarrhea, vomiting, and gastro-intestinal inflammation. Considering its occurrence in food and feedstuff, deoxynivalenol (DON) is one of the most important trichothecenes. We report the synthesis of stable isotope labeled 15-d(1)-deoxynivalenol (15-d(1)-DON) from its natural precursor 3-acetyldeoxynivalenol (3-AcDON) as starting material. Furthermore, a method for the analysis of DON and 3-AcDON using HPLC-MS/MS with stable isotope labeled 15-d(1)-DON and 3-d(3)-AcDON as internal standards has been developed. In total, 18 cereal product samples were analyzed with contamination levels ranging from 10-301 microg/kg for DON and 5-14 microg/kg for 3-AcDON. This is the first report of an isotope dilution MS method for the analysis of type B-trichothecenes.     

Analysis of sphingolipids in potatoes (Solanum tuberosum L.) and sweet potatoes (Ipomoea batatas (L.) Lam.) by reversed phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS)

Ceramides and glucocerebrosides of potatoes (Solanum tuberosum L.) and sweet potatoes (Ipomoea batatas (L.) Lam.) were analyzed using RP-HPLC-ESI-MS/MS. Ceramides and glucocerebrosides containing the three different long-chain bases 4,8-sphingadienine (d18:2(delta4,delta8)), 4-hydroxy-8-sphingenine (t18:1(delta8)), and 8-sphingenine (d18:1(delta8)) acylated to saturated and unsaturated hydroxy- and nonhydroxy fatty acids with 16-26 carbon atoms were detected. For ceramides and glucocerebrosides 4,8-sphingadienine (d18:2(delta4,delta8)) was found as the major long-chain base, with lesser amounts of 4-hydroxy-8-sphingenine (t18:1(delta8)) and 8-sphingenine (d18:1(delta8)). 2-(Alpha-)hydroxypalmitic acid (C16:0h) was the major fatty acid, which was found to be acylated to the long-chain bases. For quantification of these compounds, an RP-HPLC-ESI-MS/MS method with an "echo-peak"-technique simulating internal standard injection was developed. The analyzed samples of potatoes and sweet potatoes showed amounts of approximately 0.1-8 microg/kg single ceramides and amounts up to 500 microg/kg glucocerebrosides, with C16:0h-glucosyl-4,8-sphingadienine as the major component.     

Zur Desoxydation flüssigen Eisens mit Zirkon

An Eisenschmelzen mit 0,007% C und 0,16% O Untersuchung der Kinetik des Desoxydationsvorganges mit Eisen-Zirkon-Legierungen. Einfluß des Zirkongehaltes des Desoxydationsmitteis auf den Reaktionsablauf. Deutung der Versuchsergebnisse mit Hilfe der Keimbildungstheorie.     

Acoustic startle threshold of the albino rat (Rattus norvegicus).

The startle threshold of the albino Sprague-Dawley rat runs parallel to the curve of the hearing threshold. The difference between the startle and hearing threshold is 87 dB (SPL) at a background noise level of 75 dB (SPL). At 110 dB (SPL), the threshold has a range from 2 kHz to 50 kHz with a minimum at 10 kHz and a second minimum at 40 kHz. Amplitude and latency of the startle response are not only dependent on the sensation level of the acoustic stimulus but also on the frequency. At threshold, only the head movement component of the startle response is elicited. (PsycINFO Database Record (c) 2010 APA, all rights reserved)